Gut content examination and trophic markers (fatty acids, stables isotopes of C and N) were combined to delineate the diet of the dominant species of amphipods from Mediterranean Posidonia oceanica ... [more ▼]

Gut content examination and trophic markers (fatty acids, stables isotopes of C and N) were combined to delineate the diet of the dominant species of amphipods from Mediterranean Posidonia oceanica seagrass meadows and to highlight trophic diversity among this community. Our results indicate that, although all dominant species heavily relied on macroalgal epiphytes, considerable interspecific dietary differences existed. Carbon stable isotope ratios notably showed that some of the amphipod species favored grazing on epiphytes from leaves or litter fragments (Apherusa chiereghinii, Aora spinicornis, Gammarus aequicauda), while others like Dexamine spiniventris preferred epiphytes from rhizomes. The remaining amphipods (Caprella acanthifera, Ampithoe helleri and Gammarella fucicola) readily consumed both groups. In addition, SIAR modeling suggested that most species had a mixed diet, and relied on several food items. Fatty acid analysis and gut contents revealed that contribution of microepiphytic diatoms and of benthic and suspended particulate organic matter to the diet of amphipods were anecdotal. None of the examined species seemed to graze on their seagrass host (low 18:2(n-6) and 18:3(n-3) fatty acids contents), but G. aequicauda partly relied on seagrass leaf detritus, as demonstrated by the lesser 13C-depletion of their tissues. Overall, our findings suggest that amphipods, because of their importance in transfers of organic matter from primary producers and detritus to higher rank consumers, are key-items in P. oceanica associated food webs. [less ▲]

A feature of the organ of Corti’s supporting cells is the presence of an abundant cytoskeleton which is mainly composed of microtubules. These supporting cells have also been shown to contain a minor ... [more ▼]

A feature of the organ of Corti’s supporting cells is the presence of an abundant cytoskeleton which is mainly composed of microtubules. These supporting cells have also been shown to contain a minor mammalian tubulin, the β5-tubulin [1], recently related as a biomarker for cancer outcome [2] and cell proliferation [3]. It was shown that a β-tubulin isoform can specified the microtubule architecture, such as the expression of the Moth β2 in the Drosophila testes imposed the 16 protofilaments (16pf) structure on the corresponding subset of Drosophila microtubules, which normally contain 13pf [4]. Moreover, supporting cell microtubules are formed by 15pf instead of the canonical 13, a unique fact among vertebrates [5]. Such a protofilament configuration has been observed in C. elegans’ neurons which are responsible for the mechanosensory sense of touch [6]. It was also shown that these 15pf microtubules were essential to the proper functioning of these mechanosensory neurons [6]. To determine the role of this particular tubulin in the auditory organ and its possible involvement in the formation of the unusual 15pf microtubules of supporting cells, we studied the spatiotemporal localization of β5-tubulin during development in rats from embryonic day 18 until P25 (25th postnatal day). We also analyzed the localization of β5-tubulin mRNA expression in the Corti’s organ. Then we examined the fine structure of microtubules at the electron microscope level. For these experiments, we used an early postnatal stage and a late postnatal stage. Our results showed that β5-tubulin, contrary to other β-tubulins, had a unique distribution in the cochlea. This β-tubulin appeared at a postnatal stage, before the opening of the Corti’s tunnel and being restricted to supporting cells, especially in pillar and Deiters cells,. The same localization of β5-tubulin mRNA was observed by in Situ Hybridization. Electron microscopy indicated further that Pillar and Deiters cells were composed by 15-protofilament microtubules at the late postnatal stage (P25). In conclusion, all these data strongly suggest that there is a relationship between the presence of β5-tubulin and 15-protofilament microtubules in the supporting cells of the auditory organ. Further studies are now needed to elucidate their role. [less ▲]

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains ... [more ▼]

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. [less ▲]

Contrary to the highly specialized epithelial cells of the mammalian auditory organ, little is known about the surrounding cells and, in particular, Boettcher's cells (BC). Our morphological studies show ... [more ▼]

Contrary to the highly specialized epithelial cells of the mammalian auditory organ, little is known about the surrounding cells and, in particular, Boettcher's cells (BC). Our morphological studies show that, in rats, these cells began their differentiation around postnatal day 8 (P8) reaching maturity around P20, when they are completely covered by Hensen's and Claudius' cells. Tight junctions were noted near the apex of BC, providing that they were in direct contact with the endolymphatic space, between approximately P8 and P16. We observed gap junctions between BC and adjacent cells before the end of the covering process suggesting the additional involvement of BC in potassium recycling into the endolymph. Adherens junctions were also seen between BC throughout their maturation. Importantly, we noticed cytoplasmic secretory granules and an accumulated material, probably a secretion, in the intercellular space, between P8 and P25. These results indicate that BC could basally take part in the secretion of the extracellular matrix of the basilar membrane. Finally, we show that the basolateral interdigitations of BC are longer and more tighlty grouped at maturity and harbour urea transporters as early as P18. Our observations thus support the view that BC perform several functions. [less ▲]

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation ... [more ▼]

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. Methods: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfermethod. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanismbymite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. Results: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. Conclusions: Der p 1 in either its recombinant formor in the natural context of house dustmite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. General significance: This finding suggests that Der p 1 may be valuable target against mites. [less ▲]

In eutherian mammals, the organ responsible for the transduction of sound waves into nerve impulses is called the organ of Corti. This structure located within the cochlea, a portion of the inner ear, is ... [more ▼]

In eutherian mammals, the organ responsible for the transduction of sound waves into nerve impulses is called the organ of Corti. This structure located within the cochlea, a portion of the inner ear, is composed by two types of cells: sensory hair cells and non-sensory supporting cells. All these cells are distributed according to a specific arrangement along the whole length of the cochlea. A feature of the organ of Corti’s supporting cell is the presence of an abundant cytoskeleton. This one is mainly composed of microtubules, structures make up by tubulin heterodimers. The heterodimers consist of one molecule of α tubulin and one molecule of β tubulin. β tubulin isotypes are highly conserved in evolution and differ by only a few amino acid residues, implying that the isotypes may have functional significance. Organ of Corti’s supporting cells are constituted by class V β-tubulin, a minor mammalian tubulin (Bhattacharya et al., 2008). Moreover, their microtubules are formed by 15 protofilaments instead of the canonical 13, a unique fact among vertebrates (Banerjee et al., 2008). Such a configuration of protofilaments has been observed in C. elegans’ neurons which are responsible for the mechanosensory sense of touch (Bounoutas et al., 2009). It was also shown that these 15 protofilaments microtubules were essential to the proper functioning of these mechanosensory neurons (Bounoutas et al., 2009). Here we present the spatiotemporal localization of class V β-tubulin during the development of the organ of Corti in rats from embryonic day 18 (E18) until P25 (25th postnatal day). For this purpose, we have used immunolabelings on cryosections of whole cochlea. Our preliminary results demonstrate that class V β-tubulin has a unique distribution in the cochlea, being restricted to supporting cells, especially in pillar cells. [less ▲]

Histochemistry and cytochemistry are important fields for studying the inner workings of cells and tissues of the body. While visualization techniques have been in use for many years, new methods of ... [more ▼]

Histochemistry and cytochemistry are important fields for studying the inner workings of cells and tissues of the body. While visualization techniques have been in use for many years, new methods of detection developed recently help researchers and practitioners better understand cell activity. Histochemical and Cytochemical Methods of Visualization describes the essential techniques that can be used for histochemical investigations in both light and transmission electron microscopy. The book begins by discussing techniques in light microscopy. It reviews classical methods of visualization, histochemical and histoenzymatic methods, and methods used to visualize cell proliferation and apoptosis. Next, the book examines the cytochemical methods used in electron microscopy with traditional techniques, as well as more specialized methods. The final section provides an overview of image analysis and describes how image processing methods can be used to extract vital information. A 16-page insert supplies color illustrations to enhance the text. Techniques will continue to adapt to the latest technological innovations, allowing more and more precise quantification of images. These developments are essential to the biological as well as the medical sciences. This manual is a critical resource for novice and experienced researchers, technicians, and students who need to visualize what happens in the cell, the molecules expressed, the main enzymatic activities, and the repercussions of the molecular activities upon the structure of the cells in the body. [less ▲]

In eutherian mammals, the organ responsible for the transduction of sound waves into nerve impulses is called the organ of Corti. This structure located within the cochlea, a portion of the inner ear, is ... [more ▼]

In eutherian mammals, the organ responsible for the transduction of sound waves into nerve impulses is called the organ of Corti. This structure located within the cochlea, a portion of the inner ear, is composed by two types of cells: sensory hair cells and non-sensory supporting cells. All these cells are distributed according to a specific arrangement along the whole length of the cochlea. So far, the mammalian inner ear is very sensitive to damage, with no hair cell replacement or cell proliferation occurring in the cochlea. That is why understanding the mechanisms that regulate the mammalian cochlear development is important for pursuing strategies to induce sensory hair cells regeneration. Here, we present a technique of whole embryonic inner ear culture in rotating bioreactors. Besides, we compare two different culture media, DMEM and Neurobasal-A. Rat inner ears are sampled at the 16th embryonic day (E16) and grown in rotating bioreactors during 48h or six days. After 48h, semithin sections realized in the growing cochlea show the development of the ventral epithelium and ultrathin sections confirm the differentiation of the sensory hair cells. Using immunochemistry techniques on our material after 48h or six days in vitro, we show that all the cells of the organ of Corti are differentiating, whichever the culture medium used. Our preliminary results demonstrate that organ culture of the embryonic inner ear in rotating bioreactor is possible. Such a method provides an in vitro model for the investigation of developmental, regulatory, and differentiation processes that could be helpful in the understanding of the mechanisms underlying the development of the mammalian cochlea. [less ▲]

An epithelial-mesenchymal transition is a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. During ... [more ▼]

An epithelial-mesenchymal transition is a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. During this process, epithelial cells loosen cell-cell adhesion, module their polarity and rearrange their cytoskeleton: intermediate filaments typically switch from cytokeratin to vimentin. They also enhance their motility capacity. The epithelial-mesenchymal transition plays key roles in the formation of the body plan and in the differentiation of multiple tissues and organs but it is also involved in tissue repair, tissue homeostasis, fibrosis, and carcinoma progression. Until now, epithelial-mesenchymal transition has been rarely mentioned in the inner ear organogenesis. In chick, epithelial-mesenchymal transition has been reported as a possible mechanism of semicircular canal morphogenesis. More recently, an in vitro study has also indicated that sensory epithelial cells from mouse utricle can undergo an epithelial-mesenchymal transition to become cells expressing features of prosensory cells. By contrast, epithelial-mesenchymal transition has never been observed during auditory organ morphogenesis. The auditory organ, the organ of Corti, is a highly specialized structure composed by specific cellular types. The sensory cells are characterized by stereocilia at their apex and are necessary for the sound perception. Theses cells are supported by supporting cells. Based on their morphology and physiology, at least four types of supporting cells can be identified in the organ of Corti: inner and outer pillar cells, phalangeal cell and Deiter’s cells. The inner pillar cells and outer pillar cells combine to form the tunnel of Corti, a fluid filled triangular space that separates the single row of inner hair cells from the first row of outer hair cells. The Nuel spaces are another interval in the organ of Corti that is situated between the outer pillar cells and the different rows of outer hair cells and Deiters cells. To determine whether an epithelial-mesenchymal transition may play a role in the morphogenesis of the auditory organ, we studied the spatial localization of several epithelial-mesenchymal transition markers, the cell-cell adhesion molecules and intermediate filament cytoskeletal proteins, in epithelium of the dorsal cochlea during development of the rat organ of Corti from 18th embryonic day until 25th postnatal day. We examined by confocal microscopy immunolabelings on cryosections of whole cochleae with antibodies anti-cytokeratins as well as with antibodies anti-vimentin, anti-E-cadherin and anti-beta-catenin.Our results showed a partial loss of E-cadherin and beta-catenin between supporting cells at P8 and P12, respectively, and a temporary appearance of vimentin in pillar cells and Deiters between P8 and P10. Our results show a local loss of adhesion between supporting cells of the OC from P8, an increase expression of cytokeratins in supporting cells around P10 and a temporary appearance of vimentin in supporting cells at P8-10. These observations suggest that a partial epithelial-mesenchymal transition might be involved in the remodeling of the Corti organ during the postnatal stages of development in rat. [less ▲]

The Boettcher cells (BC) lie on the sensory epithelium of the cochlea. Their function has never been clearly defined. However it has been suggested that they may influence the ionic composition of the ... [more ▼]

The Boettcher cells (BC) lie on the sensory epithelium of the cochlea. Their function has never been clearly defined. However it has been suggested that they may influence the ionic composition of the fluids of the inner ear, which play a central role in the conduction of the sensory information. In this context the compartimentating function of the BC around and after the onset of hearing may influence the subsequent refining of hearing. We collected ultrastructural and immunohistological data during the final maturation stage of the sensory epithelium. In particular the cell junctions were investigated to clarify the compartimentating function of the BC at early stages. As a potential actor in the ion flow in the sensory epithelium, the urea transporter-B (UT-B) was also immunolocalised during the development of the BC. At the mature stage (P25) the BC are linked to the adjacent cells by numerous adherens and non-adherens junctions. They rest on a basilar membrane to which they are attached by hemidesmosomes. They typically exhibit large basolateral interdigitations. We found that, at the 8th postnatal day, the BC are separated from the neighbouring cells by wide spaces entered by scarce cytoplasmic extensions. These spaces are interrupted by areas of close contact, where adherens and non-adherens junctions may be found. Thus, although there seems to be fewer interdigitations at P8, gap junctions probably still allow easy cell-to-cell exchanges. Moreover non-adherens junctions can systematically be identified apically. Although it was impossible to differenciate tight and gap junctions without specific labeling, we postulate that these non-adherens junctions correspond to tight junctions and seal the apex of the BC. This feature is necessary to enable the control of the ion concentrations surrounding the sensory epithelium. We also found that UT-B, known for water and urea transport in red blood cells, is present in the membranes of the BC from P12 (the earliest stage tested) to P25. Thus UT-B may play a role in the regulation of the ionic concentrations of the inner ear fluids. [less ▲]

Mediterranean Posidonia oceanica meadows shelter a high biomass and an important biodiversity of amphipod crustaceans. In other temperate meadows, the amphipods play an important part in the functioning ... [more ▼]

Mediterranean Posidonia oceanica meadows shelter a high biomass and an important biodiversity of amphipod crustaceans. In other temperate meadows, the amphipods play an important part in the functioning of the ecosystem, notably in organic matter transfers from producers to higher level consumers. However, the situation in Posidonia oceanica meadows remains unclear, and little is known about the trophic ecology of amphipods, which are generally regarded as generalist herbivores/detritivores despite the lack of precise studies. Here, we combined gut content examination and trophic markers (fatty acids, stables isotopes of C and N) to delineate the diet of the dominant species of amphipods from Posidonia oceanica seagrass meadows and to highlight trophic diversity among this community. Our results indicate that contribution of microepiphytic diatoms and of benthic and suspended particulate organic matter to the diet of amphipods were anecdotal. On the other hand, all dominant species heavily relied on macroalgal epiphytes, suggesting a certain extent of overlapping in the diets of the dominant species. Considerable interspecific differences nonetheless existed, notably concerning grazing preferences towards epiphytes from leaves or litter fragments vs. epiphytes from rhizomes. In addition, the use of the SIAR isotopic mixing model showed that most species had a mixed diet, and relied on several food items. None of the examined species seemed to graze on their seagrass host, but Gammarus aequicauda partly relied on seagrass leaf detritus. Overall, our findings demonstrate that amphipods have the potential to be key-items in trophic and functional interactions occurring among Mediterranean Posidonia oceanica meadows. [less ▲]

The epithelial-mesenchymal transition (EMT) plays a crucial role in the differentiation of many tissues and organs. So far, an EMT was not detected in the development of the auditory organ. To determine ... [more ▼]

The epithelial-mesenchymal transition (EMT) plays a crucial role in the differentiation of many tissues and organs. So far, an EMT was not detected in the development of the auditory organ. To determine whether an EMT may play a role in the morphogenesis of the auditory organ, we studied the spatial localization of several EMT markers, the cell-cell adhesion molecules and intermediate filament cytoskeletal proteins, in epithelium of the dorsal cochlea during development of the rat Corti organ from E18 (18th embryonic day) until P25 (25th postnatal day). We examined by confocal microscopy immunolabelings on cryosections of whole cochleae with antibodies anti-cytokeratins as well as with antibodies anti-vimentin, anti-E-cadherin and anti-β-catenin. Our results showed a partial loss of E-cadherin and β-catenin and a temporary appearance of vimentin in pillar cells and Deiters between P8 and P10. These observations suggest that a partial EMT might be involved in the remodelling of the Corti organ during the postnatal stages of development in rat. [less ▲]

An epithelial-mesenchymal transition is a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. During ... [more ▼]

An epithelial-mesenchymal transition is a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. During this process, epithelial cells loosen cell-cell adhesion, module their polarity and rearrange their cytoskeleton: intermediate filaments typically switch from cytokeratin to vimentin. They also enhance their motility capacity. The epithelial-mesenchymal transition plays key roles in the formation of the body plan and in the differentiation of multiple tissues and organs but it is also involved in tissue repair, tissue homeostasis, fibrosis, and carcinoma progression. Until now, epithelial-mesenchymal transition has been rarely mentioned in the inner ear organogenesis. In chick, epithelial-mesenchymal transition has been reported as a possible mechanism of semicircular canal morphogenesis. More recently, an in vitro study has also indicated that sensory epithelial cells from mouse utricle can undergo an epithelial-mesenchymal transition to become cells expressing features of prosensory cells. By contrast, epithelial-mesenchymal transition has never been observed during auditory organ morphogenesis. The auditory organ, the organ of Corti, is a highly specialized structure composed by specific cellular types. The sensory cells are characterized by stereocilia at their apex and are necessary for the sound perception. Theses cells are supported by supporting cells. Based on their morphology and physiology, at least four types of supporting cells can be identified in the organ of Corti: inner and outer pillar cells, phalangeal cell and Deiter’s cells. The inner pillar cells and outer pillar cells combine to form the tunnel of Corti, a fluid filled triangular space that separates the single row of inner hair cells from the first row of outer hair cells. The Nuel spaces are another interval in the organ of Corti that is situated between the outer pillar cells and the different rows of outer hair cells and Deiters cells. To determine whether an epithelial-mesenchymal transition may play a role in the morphogenesis of the auditory organ, we studied the spatial localization of several epithelial-mesenchymal transition markers, the cell-cell adhesion molecules and intermediate filament cytoskeletal proteins, in epithelium of the dorsal cochlea during development of the rat organ of Corti from 18th embryonic day until 25th postnatal day. We examined by confocal microscopy immunolabelings on cryosections of whole cochleae with antibodies anti-cytokeratins as well as with antibodies anti-vimentin, anti-E-cadherin and anti-beta-catenin.Our results showed a partial loss of E-cadherin and beta-catenin between supporting cells at P8 and P12, respectively, and a temporary appearance of vimentin in pillar cells and Deiters between P8 and P10. Our results show a local loss of adhesion between supporting cells of the OC from P8, an increase expression of cytokeratins in supporting cells around P10 and a temporary appearance of vimentin in supporting cells at P8-10. These observations suggest that a partial epithelial-mesenchymal transition might be involved in the remodeling of the Corti organ during the postnatal stages of development in rat. [less ▲]

The auditory organ, the organ of Corti (OC), is a highly specialized structure composed by specific cellular types. The sensory cells (HC) are characterized by stereocilia at their apex and are necessary ... [more ▼]

The auditory organ, the organ of Corti (OC), is a highly specialized structure composed by specific cellular types. The sensory cells (HC) are characterized by stereocilia at their apex and are necessary for the sound perception. Theses cells are supported by supporting cells. Based on their morphology and physiology, at least four types of supporting cells (SC) can be identified in the OC: inner and outer pillar cells (PC), phalangeal cell and Deiter’s cells. Sensory and supporting cells possess characteristic cytoskeleton proteins in direct relation with their morphological features and their development. Indeed, this organ had morphological changes such as the setting up of the sensory epithelium after the birth or the openings of the Corti’s tunnel at P8 and of the Nuel’s spaces at P10. In the present study, by using confocal microscopy, we investigated the spatio-temporal localization of the three cellular cytoskeletal filaments : microtubules (β-1, 2, 3, 4-tubulin), microfilaments (cytoplasmic β- and γ-actin) and intermediate filaments (CK4, 5, 7, 8, CKpan and vimentin) during the development of the OC in rat from the embryonic day 18 (E18) to the post-natal day 25 (P25). The immunolabellings indicated clearly that β-1, 2, 3-tubulins were only present the SC and nervous fibers during development whereas β-4-tubulin was found firstly in the HC and then in the SC. The two actin-isotypes were detected in the HC apex but were also seen in the PC from P8 to P25 for β-actin isoform and in the basal membrane from E18 to P8 for the γ-actin isoform. All intermediate filament proteins were only found in the SC, especially between P8 and P12. Our results show that the localization of the cytoskeleton proteins during the auditory organ development depends on the cellular type and the developmental stage. A profound modification of cytoskeleton occurs between P8 and P12. [less ▲]